The complete amino acid sequence of a vertebrate nonmuscle myosin heavy chain-B isoform (MHC-B; 1976 amino acids, 229 kDa) was deduced by using cDNA clones from chicken brain libraries. The chicken nonmuscle MHC-B shows overall similarity in primary structure to other MHCs in the areas contributing to the ATP binding site and actin binding site. Similar to other nonsarcomeric MHC IIs, there is a short uncoiled tail sequence at the carboxyl terminus of the molecule. It is in the uncoiled tail sequence that the greatest number of differences in amino acids sequence between MHC-A and B were found, which allowed generation of isoform- specific antibodies. These antibodies were used to determine the relative content of MHC-A and MHC-B in various tissues and showed that, whereas MHC-A was predominant in adrenal, kidney and spleen, MHC-B was predominant in tissues derived from the nervous system. During the cloning of the cDNA encoding chicken brain MHC-B, we found a 63 nucleotide insertion encoding 21 amino acids located in the head region of the MHC near to the actin-binding site and a 30 nucleotide insertion encoding 10 amino acids near to the ATP binding site. Analysis using S-1 nuclease showed that both inserts are expressed in a tissue-dependent manner; mRNA containing the inserts is present in tissues of the nervous system, but is absent from other nonmuscle cells, which contain the noninserted isoform of MHC-B. Similar inserts were found in corresponding positions in human cerebellar mRNA. Of note was the presence of a cdc2 kinase site in the sequence located near to the ATP binding site. A peptide based on this sequence was found to be a substrate for this kinase. Antibodies raised against a peptide synthesized based on the 21 amino acid insert found in chickens recognize a MHC isoform in the same tissues that are enriched for the mRNA. These insertions appear to be a mechanism for generating additional MHC-B isoforms specific to the nervous system. Moreover, since they are located near to the ATP and actin binding sites, they may play an important role in regulating the actin-activated MgATPase activity of myosin.